PEG-Urate Oxidase Conjugates and Use Thereof

Abstract

A naturally occurring or recombinant urate oxidase (uricase) covalently coupled to poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), wherein an average of 2 to 10 strands of PEG are conjugated to each uricase subunit and the PEG has an average molecular weight between about 5 kDa and 100 kDa. The resulting PEG-uricase conjugates are substantially non-immunogenic and retain at least 75% of the uricolytic activity of the unmodified enzyme.

1 - 41 . (canceled) 
     
     
         42 . An isolated tetrameric uricase produced by a method comprising
 applying a solution of uricase to at least one separation column at a pH of 9 to 10.5; and   recovering from said column one or more fractions that contain isolated tetrameric uricase, wherein said one or more fractions are substantially free of uricase aggregates.   
     
     
         43 . The isolated tetrameric uricase of  claim 42 , wherein the uricase is a mammalian uricase or a recombinant form thereof. 
     
     
         44 . The isolated tetrameric uricase of  claim 42 , wherein the uricase is a fungal or microbial uricase or a recombinant form thereof. 
     
     
         45 . The isolated tetrameric uricase of  claim 44 , wherein the fungal or microbial uricase is isolated from  Aspergillus flavus, Arthrobacter globiformis , or  Candida utilis , or is a recombinant enzyme having substantially the sequence of one of those uricases. 
     
     
         46 . The isolated tetrameric uricase of  claim 42 , wherein the uricase is an invertebrate uricase or a recombinant form thereof. 
     
     
         47 . The isolated tetrameric uricase of  claim 46 , wherein the invertebrate uricase is isolated from  Drosophila melanogaster  or  Drosophila pseudoobscura , or is a recombinant enzyme having substantially the sequence of one of those uricases. 
     
     
         48 . The isolated tetrameric uricase of  claim 42 , wherein the uricase is a plant uricase or a recombinant form thereof. 
     
     
         49 . The isolated tetrameric uricase of  claim 48 , wherein the plant uricase is isolated from root nodules of  Glycine max  or is a recombinant enzyme having substantially the sequence of that uricase. 
     
     
         50 . The isolated tetrameric uricase of  claim 42 , each subunit of the uricase being subsequently covalently linked to an average of 2 to 12 strands of PEG to form a PEG-uricase conjugate, wherein each molecule of PEG has a molecular weight of 5 kDa to 100 kDa. 
     
     
         51 . The isolated tetrameric uricase of  claim 50 , wherein the PEG has an average molecular weight of 10 kDa to 60 kDa. 
     
     
         52 . The isolated tetrameric uricase of  claim 51 , wherein the PEG has an average molecular weight of 20 kDa to 40 kDa. 
     
     
         53 . The isolated tetrameric unease of  claim 50 , wherein the average number of covalently coupled strands of PEG is 2 to 10 strands per uricase subunit. 
     
     
         54 . The isolated tetrameric uricase of  claim 53 , wherein the average number of covalently coupled strands of PEG is 3 to 8 strands per uricase subunit. 
     
     
         55 . The isolated tetrameric uricase of  claim 54 , wherein the average number of covalently coupled strands of PEG is 4 to 6 strands per uricase subunit. 
     
     
         56 . The isolated tetrameric uricase of  claim 50 , wherein the strands of PEG are covalently coupled to uricase via linkages selected from the group consisting of urethane linkages, secondary amine linkages, and amide linkages. 
     
     
         57 . The isolated tetrameric uricase of  claim 50 , wherein the PEG is linear. 
     
     
         58 . The isolated tetrameric uricase of  claim 50 , wherein the PEG is branched.